The study plan was reviewed and approved by the institutional review board of Severance Hospital in the Yonsei University Health System, Seoul, Korea and the trial was registered at www.clinicaltrials.gov (the clinical trial registry number is NCT01455558).
Pharmacokinetics
Six separate clinical trials comparing the PK profiles between newly developed sustained release (SR) formulations and an immediate release (IR) formulation of cilostazol in healthy Korean participants were completed at the Yonsei University Severance Hospital (Seoul, Korea) from August 2008 to August 2009, however the data only from IR formulation were used in this study [17]. In brief, each participant received two IR (100 mg × two tablets, BID) formulations of cilostazol (PLETAAL), with one tablet administered every 12 h. Venous blood samples were collected into heparin tubes at 0, 1, 2, 3, 4, 6, 8, 10, 12, 13, 14, 15, 16, 18, 20, 22, 24, 36, 48, and 72 h after drug administration. The plasma concentrations of cilostazol and its metabolites (OPC-13015 and OPC-13213) were measured by using liquid chromatography tandem mass spectrometry (LC-MS/MS), and PK profiles, including the parameters Cmax, AUCinf, and t1/2, were computed by using Phoenix WinNonlin version 5.3 (Pharsight Corporation, Mountain View, CA).
Participants
The volunteers who had participated in this clinical trials were healthy Korean males or females between 19 and 55 years of age, within 20% of their ideal body weight, and without congenital abnormalities or chronic diseases (17). Informed consent for the analysis of their genetic information was obtained from all 101 participants. In total, 33 volunteers that gave consent for MRP5 mRNA analysis were newly recruited, in accordance with the same criteria as those of clinical trials, because this study using clinical trial data was planned retrospectively.
Adverse Drug Reactions
Basic vital signs (blood pressure, body temperature, and pulse rate), a personal symptom interview, electrocardiograms (ECGs), pregnancy tests (human chorionic gonadotropin in blood) for females, and laboratory tests (hematology, blood chemistry, and urinalysis) were performed at appropriate times. An ADR was defined as any unfavorable result in the checked information and was recorded on the case-report forms [17]. All participants who had enrolled in the clinical trials were questioned about headaches by using the 10-point VRS during a personal interview. The headache intensity was categorized into four groups, no headache (0), mild headache (1–3), moderate headache (4–6), and severe headache (7–10) according to the numeric rating scale (NRS) used in an earlier report (18). For statistical analysis, the four groups were re-categorized into two groups: no or mild headache (0–3), and moderate or severe headache (4–10).
gDNA, RNA extraction, and cDNA synthesis
gDNA was extracted from fresh human whole blood by using ExgeneTM Blood SV mini kit (GeneAll, Seoul, Korea) in accordance with the manufacturer’s instructions. Leukocytes were obtained from fresh human whole blood using erythrocyte lysis buffer (0.15 M NH4Cl, 10 mM KHCO3, 0.1 mM EDTA). Total RNA was extracted from leukocytes by using TRIzol Reagent (Invitrogen, Carlsbad, CA), and RNA was converted into cDNA using an AccuScript High Fidelity 1st-Strand cDNA Synthesis Kit (Stratagene, Santa Clara, CA) in accordance with the manufacturer’s instructions.
Gene scanning and genotyping
A promoter region of −2.0 kb from the downstream transcription start site, all exons in their entirety, and the flanking intronic sequences of the MRP4 and MRP5 genes were scanned by a DNA direct sequencing method using the ABI Prism 3730xl Genetic Analyzer (Applied Biosystems, Foster City, CA) in accordance with the manufacturer’s instructions. Mutations were analyzed using Phred, Phrap, Consed, and Polyphred 5.04 software (http://droog.mbt.washington.edu/PolyPhred.html). Haplotype and LD structure were constructed by using the Haploview software package (version 4.0; Broad Institute of MIT and Harvard, Cambridge, MA). SNPs were genotyped with the SNaPshot assay using the ABI Prism 3730xl Genetic Analyzer, and the results were analyzed by using GeneScan analysis software (Applied Biosystems).
Allele quantification for c.1146A>G of MRP5
The allele quantification ratio was compared between the A and G alleles at c.1146A>G of MRP5 gDNA and cDNA using the PyroMark Q24 system (Qiagen, Valencia, CA) in accordance with the manufacturer’s instructions (sequencing primer for gDNA: 5′-CATTTTCTCAGAGTGTTCA-3′, for cDNA: 5′-CTCCTCGCGGATTTT-3′).
The following primers were used for PCR: Forward primer for gDNA, 5′-ATCAAAATGTATGCCTGGGTCAAA-3′, reverse primer for gDNA (5′ biotinylated): 5′-AAAGAATCCAAGGTCCTTCTACCA-3′; forward primer for cDNA (5′ biotinylated): 5′-TGCCTGGGTCAAAGCATTTT-3′, reverse primer for cDNA: 5′-ATCGAAGCCCAGGGTCAT-3′. PCR was conducted using the PyroMark PCR Kit (Qiagen) by following cycling conditions: 15 min at 95°C; 45 cycles of 30 s at 94°C; 30 s at 56°C; and 30 s at 72°C; followed by a final cycle at 72°C for 10 min.
Cell management
HEK cells were maintained at MEM (LM007-07, Welgene) with 10% FBS (16000044, Gibco) and 1% Penicillin-streptomycin solution (LS202-02, Welgene) in the humidified 5% CO2 incubator.
Immunoblotting
HEK293 cells (1×106 cells) were seeded in 60 mm plates at the day before transfection and then transfected for 36 h by using Lipofectamine 2000. The cells were washed with ice-cold phosphate-buffered saline (PBS), lysed in radioimmunoprecipitation assay (RIPA) lysis buffer containing 0.5% sodium dodecyl sulfate (SDS), 1% Nonidet P-40, 1% sodium deoxycholate, 150 mM Tris-hydrochloride (HCl, pH 7.4), and protease inhibitors. The protein concentration was determined by using Bio-Rad protein assay dye reagent. The proteins were separated by using SDS-polyacrylamide gel electrophoresis (PAGE) in a 4%–12% gradient gel (Invitrogen) and transferred to PVDF membranes (Amersham Biosciences). Non-specific binding of protein to the membranes was blocked by 5% nonfat skim milk for 1 h incubation at 37°C. The membranes were washed three times (each of 10 min) with tris-buffered-saline (TBS) containing tween 20 (0.1 %) and probed with anti-MRP5 (1:500, sc-20769, Santa Cruz Biotechnology), anti-beta actin antibodies (1:10,000, A5441, Sigma) for 30 min at 37°C. The membranes were washed three times again as mentioned before and incubated with horseradish-peroxidase-conjugated anti-mouse IgG secondary antibody (1:2,000, Santa Cruz) for 2 h at room temperature. The membranes were then exposed to a medical x-ray film using the enhanced chemiluminescence (ECL) system (Santa Cruz).
Immunocytochemistry
HEK293 cells (1×106 cells) were seeded in 60 mm plates containing two microscope cover glasses at the day before transfection. The cells were then transfected with cDNA constructed for wild type (A allele) and mutant (G allele) MRP5 gene by using Lipofectamine 2000. The cells were incubated for 36 h, then culture medium was removed and the cells were fixed with 4% paraformaldehyde in PBS at 4°C for 15 min. After that, paraformaldehyde was removed the cells were incubated with blocking solution (3% BSA and 1% normal horse serum in PBS) for 1 h at 37°C. The cells were then incubated with rabbit polyclonal anti-human MRP5 (1:100, sc-20769, Santa Cruz) and mouse monoclonal anti-human P-cadherin (1:100, ab6528, Abcam) in blocking solution at 37°C for 30 min. After removal of the solution containing the primary antibodies, the cells were incubated with anti-rabbit FITC (1:200, sc-2012, Santa Cruz) and anti-mouse Alexa Fluor 546 (1:200, A11003, Life Technologies) in blocking solution at room temperature for 1 h. After washing in PBS, the cells were mounted on slides and were visualized under a confocal microscope (LSM700, Carl Zeiss, Germany).
Statistical analysis
SPSS 18.0 (SPSS Inc., Chicago, IL) and GraphPad Prism 4.0 (GraphPad Software, Inc., San Diego, CA) software packages were used for statistical analyses. All genotyped SNPs underwent Hardy-Weinberg equilibrium testing before case-control analysis. Categorical data were evaluated by using the chi-square and Fisher’s exact tests, whereas ANOVA, t-test, and Kruskal-Wallis test were applied to continuous data. A significant effect of a certain genotype on the clinical phenotype was estimated as an OR with a 95% CI by using a binary logistic regression method. The fundamental standard P value of significance was based on two-sided comparisons and was selected as 0.05. However, the P value was adjusted with Bonferroni’s correction when multiple statistical analyses were performed at once.